Nigella Plants – Traditional Uses, Bioactive Phytoconstituents, Preclinical and Clinical Studies

Nigella Plants –Traditional Uses, Bioactive Phytoconstituents, Preclinical and Clinical Studies Bahare Salehi 1 , Cristina Quispe 2 , Muhammad Imran 3 , Iahtisham Ul-Haq 4 , Jelena ˇ Zivkovi ´c 5 , Ibrahim M. Abu-Reidah 6 , Surjit Sen 7 , 8 , Yasaman Taheri 9 , Krishnendu Acharya 7 , Hamed Azadi 10 , María del Mar Contreras 11 *, Antonio Segura-Carretero 12,13 , Dima Mnayer 14 , Gautam Sethi 15 , Miquel Martorell 16,17 , Ahmad Faizal Abdull Razis 18,19 *, Usman Sunusi 19,20 , Ramla Muhammad Kamal 19,21 ,Hafiz Ansar Rasul Suleria 22 and Javad Sharifi-Rad 9 ,23 * 1 Medical Ethics and Law Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran, 2 Facultad de Ciencias de la Salud, Universidad Arturo Prat, Iquique, Chile, 3 Faculty of Allied Health Sciences, University Institute of Diet and Nutritional Sciences, The University of Lahore, Lahore, Pakistan, 4 Department of Diet and Nutritional Sciences, Faculty of Health and Allied Sciences, Imperial College of Business Studies, Lahore, Pakistan, 5 Institute for Medicinal Plants Research “Dr. Josif Pan ˇci ´c”, Belgrade, Serbia, 6 Department of Environmental Science/Boreal Ecosystem Research Initiative, Memorial University of Newfoundland, Corner Brook, NL, Canada, 7 Molecular and Applied Mycology and Plant Pathology Laboratory, Department of Botany, University of Calcutta, Kolkata, India, 8 Department of Botany, Fakir Chand College, Diamond Harbour, India, 9 Phytochemistry Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran, 10 Department of Agronomy and Plant Breeding Science, College of Aburaihan, University of Tehran, Tehran, Iran, 11 Department of Chemical, Environmental and Materials Engineering, University of Jaén, Jaen, Spain, 12 Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Granada, Spain, 13 Research and Development Functional Food Centre (CIDAF), Bioregión Building, Health Science Technological Park, Granada, Spain, 14 Faculty of Sciences, Lebanese University, Beirut, Lebanon, 15 Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore, 16 Department of Nutrition and Dietetics, Faculty of Pharmacy, and Centre for Healthy Living, University of Concepción, Concepción, Chile, 17 Unidad de Desarrollo Tecnológico, UDT, Universidad de Concepción, Concepción, Chile, 18 Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, Serdang, Malaysia, 19 Natural Medicines and Products Research Laboratory, Institute of Bioscience, Universiti Putra Malaysia, Serdang, Malaysia, 20 Department of Biochemistry, Bayero University Kano, Kano, Nigeria, 21 Department of Pharmacology, Federal University Dutse, Dutse, Nigeria, 22 Department of Agriculture and Food Systems, The University of Melbourne, Melbourne, VIC, Australia, 23 Facultad de Medicina, Universidad del Azuay, Cuenca, Ecuador Nigella is a small genus of the family Ranunculaceae, which includes some popular species due to their culinary and medicinal properties, especially in Eastern Europe, Middle East, Western, and Central Asia. Therefore, this review covers the traditional uses and phytochemical composition of Nigella and, in particular, Nigella sativa. The pharmacological studies reported in vitro,in vivo, and in humans have also been reviewed. One of the main strength of the use of Nigella is that the seeds are rich in the omega-6 fatty acid linoleic acid and provide an extra-source of dietary phytochemicals, including the bioactive thymoquinone, and characteristics saponins, alkaloids, and flavonoids. Among Nigella species, N.sativa L. is the most studied plant from the genus. Due to the phytochemical composition and pharmacological properties, the seed and seed oil from this plant can be considered as good candidates to formulate functional ingredients on the basis of folklore and scientific knowledge. Nonetheless, the main limations are that more studies, especially, clinical trials are required to standardize the results, e.g. to establish active molecules, dosage, chemical profile, long-term effects and impact of cooking/incorporation into foods. Keywords: Nigella, cancer, pharmacological properties, functional ingredients, metabolic syndrome, thymoquinone Edited by: Andrei Mocan, Iuliu Hatieganu University of Medicine and Pharmacy, Romania Reviewed by: Qiang Guo, Peking University Health Science Centre, China Abdulrahman Koshak, University College London, United Kingdom *Correspondence: María del Mar Contreras [email protected] Ahmad Faizal Abdull Razis [email protected]y Javad Sharifi-Rad javad.sharifi[email protected] Specialty section: This article was submitted to Ethnopharmacology, a section of the journal Frontiers in Pharmacology Received: 02 November 2020 Accepted: 16 February 2021 Published: 26 April 2021 Citation: Salehi B, Quispe C, Imran M, Ul-Haq I, ˇ Zivkovi ´c J, Abu-Reidah IM, Sen S, Taheri Y, Acharya K, Azadi H, del Mar Contreras M, Segura-Carretero A, Mnayer D, Sethi G, Martorell M, Abdull Razis AF, Sunusi U, Kamal RM, Rasul Suleria HA and Sharifi-Rad J (2021) Nigella Plants –Traditional Uses, Bioactive Phytoconstituents, Preclinical and Clinical Studies. Front. Pharmacol. 12:625386. doi: 10.3389/fphar.2021.625386 Frontiers in Pharmacology | www.frontiersin.org April 2021 | Volume 12 | Article 6253861 REVIEW published: 26 April 2021 doi: 10.3389/fphar.2021.625386 INTRODUCTION Nigella, also known as fennel flower, is a small genus belonging to the family Ranunculaceae and includes around 20 species (Zohary, 1983;The plant list, 2020). The members of the genus are annuals and survive harsh condition as seed (therophytes) with a short life cycle (Agradi et al., 2002). A popular ornamental species, Nigella damascena L. (commonly known as lady-in-a-mist or ragged lady), and a well-known condiment and spice, Nigella sativa L. (also known as black cumin or black seeds), have a high commercial interest especially in the food, pharmaceutical and cosmetics industries (Ali and Blunden, 2003;Bittkau and Comes, 2009; Malhotra, 2012). As an example, N.sativa is used in foods, pickles, and baked goods (Malhotra, 2012). The evolutionary origins of Nigella species are presumably in the Aegean and the adjacent Western-Irano-Turanian region; its centre of species diversity (Zohary, 1983;Bittkau and Comes, 2009). The genus is found as wild in southern Europe, Russia, northern Africa, Asia Minor, Turkey, Middle-East, India, Pakistan, and Bangladesh (Tonçer and Kizil, 2004;Zohary, 1983;Dönmez and Mutlu, 2004;Heiss and Oeggl, 2005; Sharma et al., 2009;Iqbal et al., 2010;Heiss et al., 2011; Rabbani et al., 2011;Zhao et al., 2013;Hossan et al., 2018). The taxonomic position of Nigella has undergone many changes in the past few decades. It has been commonly divided into three sections viz. Komaroffia,Garidella and Nigella (Tutin et al., 1972;Tamura, 1993;U˘ gurlu Aydın and Dönmez, 2019). Section Komaroffia comprises one species (Nigella integrifolia Regel), section Garidella consists of two species (Nigella nigellastrum (L.) Willk. and Nigella unguicularis (Poir.) Spenn.), whereas section Nigella composed of twelve species, including N. sativa, N. damascena and others (Nigella arvensis L., Nigella fumariifola Kotschy, Nigella hispanica L., Nigella segetalis M. Bieb., Nigella stellaris Boiss., Nigella elata Boiss., Nigella ciliaris DC., Nigella orientalis L., Nigella oxypetala Boiss., and Nigella turcica Dönmez and Mutlu) (Zohary, 1983; Heiss et al., 2011). In the Plant List, a working list of known plant species produced by the botanical community, there are 91 Nigella names, but only 23 are accepted latin names for species (The plant list, 2020). In general, this genus is characterized by angular or discoid seeds and the characteristic black color is related to other common name of Nigella,“black cumin”(Zhao et al., 2013). Due to their ethnopharmacology as healing herbs and food importance of Nigella spp., the present work reviews their traditional uses and phytochemical composition,as well as various scientific studies related to their health benefit with special emphasis in N. sativa. TRADITIONAL USES OF NIGELLA Latest ethno-pharmacological studies showed that Nigella species are among the most usually used for traditional and folk medicinal practices. Among them, N. sativa is probably the best-known species of the Nigella genus and it has been used in many parts of the world as a natural medicine. The traditional use of N. sativa dates from the 1st century A.D.; Pliny the Elder recommended N. sativa as a digestive and an ingredient of antidotes curing snake bites and scorpion stings (Dönmez and Mutlu, 2004), or even at least till Tutankhamen kingdom (Mrozek-Wilczkiewicz et al., 2016). Today, the seed powder of N. sativa is recommended at 0.5–4 g in the Pharmacopoeia of India (Tajmiri et al., 2016), which is used as a stimulant to ease bowel and indigestion problems and as carminative. It has also been administered to manage pain during menstruation and diabetes in India and Bangladesh (Esakkimuthu et al., 2016; Hossan et al., 2018). Similarly, N. sativa is widely used in traditional medicine of Algeria for the treatment of diabetes and also to treat high blood pressure (BP) (Bouzabata, 2013). Moreover, according to the Bedouins (Egypt), the wooden stem is used to treat jaundice, while seeds are used to treat BP as before, as well as heart diseases, etc. (El-Seedietal.,2013)(Table 1). Similar uses have been reported in Iranian traditional medicine (Ghazeeri et al., 2012;Amiri and Joharchi, 2013;Bahmani et al., 2016b), in Pakistan (Khan et al., 2014;Yaseen et al., 2015;Aziz et al., 2017)and in Morocco (Eddouks et al., 2002;El-Hilaly et al., 2003;Khabbach et al., 2012;Jamila and Mostafa, 2014;Teixidor-Toneu et al., 2016), where N. sativa seeds and leaves are orally ingested, consumed as a powder, herbal tea, as decoction or as inhalant. In Pakistan N. sativa is also applied to manage lactation, bacterial diseases, etc. (Khan et al., 2014;Aziz et al., 2017), while in Morocco the seeds are recommended to deal with otolaryngological, urological, and nephrological ailments as well as to treat pathologies of the respiratory and skeleton–muscular systems, allergy and hypersensibility (Eddouks et al., 2002;El-Seedietal.,2013;Jamila and Mostafa, 2014). The anti-rheumatic and analgesic properties of N. sativa combined with honey have been reported (Khabbach et al., 2012).Inaddition,seedsinfusionisusedtotreatmalariainthe Malaysian traditional medicine (Al-Adhroey et al., 2010), as well as seeds (fresh, dried, and powdered forms) and leaves in Ethiopia (Alrawi et al., 2017). The use of N. sativa is still more widespread, being recognized as panacea for its healing properties in Qatar (Alrawi et al., 2017) and Bangladesh (Jennings et al., 2015). Some of the latter usesarealsocommoninMauritius, Nepal, Turkey, Thailand, Lebanon, and Palestine (Ghazeeri et al., 2012;Al-Ramahi et al., 2013;Sreekeesoon and Mahomoodally, 2014;Guler et al., 2015; Jennings et al., 2015;Kunwar et al., 2015;Bahmani et al., 2016a; Neamsuvan et al., 2016;Alrawi et al., 2017;Ahmed et al., 2018). Furthermore, in Bangladesh and Libano N. sativa seeds are used as a spice and food preservative, directly consumed after being ground, while Nigella oil also can be applied topically (Jennings et al., 2015). Similarly, seeds of N. sativa (also named Nigella glandulifera Freyn and Sint.) are consumed in some regions of China and frequently added to “naan”(a crusty pancake). Its water decoction is used in the Uighur’s traditional medicine for the treatment of numerous disorders as the other species (Table 1)(Zhao et al., 2013). Other Nigella species with a wide range of medicinal properties are N. damascena and N. ciliaris. In Central Europe, the use of N. damascena dates from Bronze Age but it cannot reliably assign any ethnobotanical relevance and its origin is unclear; it has never grown in the wild in central Europe (Heiss and Oeggl, 2005). Alternatively, N. damascena seeds are used for example in Sicilian folk medicine as a galactogogue (Geraci et al., 2018). Other uses Frontiers in Pharmacology | www.frontiersin.org April 2021 | Volume 12 | Article 6253862 Salehi et al. A Comprehensive Review on Nigella Plants are as emmenagogue, vermifugue, and disinfectant (Heiss and Oeggl, 2005). This plant is also used as an helmintic agent and to treat hematuria, and skin diseases in the Serbian medieval medicine (Jari´ c et al., 2014). The former use has also been reported in Epirus (Greece) (Vokou et al., 1993). Traditionally, N. damascena is used for treating trachoma in Tunisia and Italy (Leporatti and Ghedira, 2009). Besides its use as herbal remedy, N. damascena is used as a condiment in several regions (Heiss and Oeggl, 2005), including in Morocco (Khabbach et al., 2011). In the folklore medicine of Palestine and Iran, N. ciliaris seeds are used for abdominal pain, to facilitate delivery and to treat menstrual cycle related problems, respectively (Ali-Shtayeh et al., 2015) (Bahmani et al., 2015). In Turkey, Meriç Town, dried flowers of N. arvensis are used as a winter tea (Kartal and Günes ¸, 2017). On the other hand, in Iran (Kerman), the seed powder of N. arvensis mixed with other seeds are administrated to enhance male potency and to improve memory and intelligence (Khajoei Nasab and Khosravi, 2014). The seeds of both Nigella species are used for the treatment of cancer in Palestine (Jaradat et al., 2016a;Hamarsheh et al., 2017). PHYTOCONSTITUENTS Nigella genus is widely used for their culinary and medicinal properties especially in the Eastern Europe, Middle East, Western and Central Asia. The plants are mainly consumed for their seeds and seed oil, as commented before. The main constituents of N. sativa seeds reported in the literature are fixed oil (27–40%), proteins (16–19%), characterized mainly by the amino acids arginine, glutamic acid, leucine, lysine; minerals (1.79–3.74%), like Cu, Zn, P, and Fe; carbohydrates (28.5–33.7%), and solubre dietary fibers (5.5–8.9%) (Al-Naqeep et al., 2009;Tiruppur Venkatachallam et al., 2010;Kooti et al., 2016;El-Naggar et al., 2017;Saxena et al., 2017). Concerning the phytochemical composition of Nigella seeds, the most interesting plant part, it is constituted of alkaloids, terpenes and phenolic compounds (Agradi et al., 2001). Although the number of studies on the chemical composition and pharmacological properties of N. sativa is continuously increasing (Bourgou et al., 2010b), there are other interesting species for their culinary and medicinal properties, as commented before. Thus, this section details phytochemicals found in N. sativa and other Nigella plants. Fixed Oil: Essential Fatty Acids The oil of N. sativa seeds has been highly studied, showing predominance of linoleic acid (1) (50–60%), oleic acid (2) (20%), myristic acid (3) (30%), and palmitic acid (4) (12.5%) (Tiruppur Venkatachallam et al., 2010;Kooti et al., 2016;ElNaggar et al., 2017;Saxena et al., 2017)(Figure 1). Nigella seed oil from Morocco also showed that the major fatty acids were linoleic acid (1) (58.5 and 56.5%), oleic acid (2) (23.8 and 24.9%) and palmitic acid (4) (13.1 and 11.9%) using cold press and solvent extraction, respectively (Gharby et al., 2015). The major compounds were similar to those found in N. sativa seeds from other Mediterranean countries. These fatty acids were TABLE 1 | Some traditional uses of Nigella species. Species Traditional use Country References Asia N. arvensis (seed) To treat lung, brain and skin Palestine (Jaradat et al., 2016a) N. ciliaris (seed) To treat abdominal pain and to facilitate delivery Palestine (Ali-Shtayeh et al., 2015) N. ciliaris (seed) To treat menstrual cycle problems Iran (Bahmani et al., 2015) N. ciliaris (seed) To treat cancer Palestine ((Jaradat et al., 2016a;Hamarsheh et al., 2017) N. sativa (seed) To treat diuretic, analgesic, insomnia, dizziness, tinnitus, amnesia, and bronchial disorders China (Zhao et al., 2013) N. sativa (seed) To ease bowel and indigestion problems and to manage diabetes India (Tajmiri et al., 2016) (Esakkimuthu et al., 2016) N. sativa (seed) To manage pain during menstruation and diabetes Bangladesh (Esakkimuthu et al., 2016)(Esakkimuthu et al., 2016)(Hossan et al., 2018 N. sativa (seed) Curative effects in bacterial-caused diseases, sexual tonic, to manage lactation and to decrease mental disturbances Pakistan (Khan et al., 2014;Aziz et al., 2017) N. sativa (seed) To treat malaria Malaysia (Al-Adhroey et al., 2010) Africa N. sativa (seed) Hypoglycemic and hypotensive agent Algeria (Bouzabata, 2013) N. sativa (wooden stem and seed) Wooden steem: To treat jaundice. Seeds: Hypotensive agent and to treat heart diseases, headaches, nasal congestion, toothache, and against intestinal worms Egypt (El-Seedi et al., 2013) N. sativa (seed, fruit and leaf) Hypoglycemic and hypotensive agent and to deal with digestive, respiratory, and cardiovascular problems, and allergy Morocco (Eddouks et al., 2002;El-Seedi et al., 2013; Jamila and Mostafa, 2014) Europe N. damascena Galactagogue (seed) and against trachoma Italy (Geraci et al., 2018)(Leporatti and Ghedira, 2009) N. damascena Antihelmintic (for children) and to treat haematuria and skin diseases (itchiness and eczema) Serbia (Geraci et al., 2018) Frontiers in Pharmacology | www.frontiersin.org April 2021 | Volume 12 | Article 6253863 Salehi et al. A Comprehensive Review on Nigella Plants also reported as major compounds from the seed oils, extracted by n-hexane, of different N. sativa genotypes from India (Saxena et al., 2017). Other study have shown that fixed oils from N. sativa seed from Turkey and Egypt obtained by supercritical CO 2 extraction were similar in fatty acid composition of saturated fatty acids (16%), mainly constituted by palmitic acid (4) and stearic acid (5); monounsaturated acids (23%), mainly oleic acid and cis-vaccenic acid (6), and polyunsaturated acids (58%), mainly linoleic acid (1) and eicosadienoic acid (7) (Piras et al., 2013)(Figure 1). In this work the ratio omega-6/omega-3 ranged between 180 and 221 considering linoleic and alphalinolenic acid. Other Nigella species, including N. damascena,N. orientalis, N. arvensis,N. elata,N. nigellastrum,N. oxypetala,N. segetalis,N. unguicularis, and N. lancifolia Hub.-Mor., also contain linoleic acid (31.2–69.5%) and oleic acid (15.8–36.0%) as the major ones (Kökdil et al., 2005;Matthaus and Özcan, 2011). The abundance relevance of other fatty acids depends on the species. Volatile Oil Phytochemicals Volatile oils are derived from plant tissues and are characterized to evaporate under room temperature and failure to saponify. Concerning volatile oil (around 0.5–1.5%) of Nigella seeds, it showed high abundance of thymoquinone (8), p-cymene (9) and other phenolic derivatives, such as dithymoquinone (nigellone) (10), thymohydroquinone (11), carvacrol (12), and thymol (13) (Bourgou et al., 2010b;Tiruppur Venkatachallam et al., 2010; Kooti et al., 2016;El-Naggar et al., 2017). These compounds are considered Nigella active principles (Mahmoudvand et al., 2014), but the composition may vary depending on the chemotype and species, among other factors (Edris, 2010;Zribi et al., 2014;Koshak et al., 2017;Saxena et al., 2017). As an example, N. sativa volatile composition (seeds) presents different chemotypes (Burits and Bucar, 2000;Islam et al., 2004): thymoquinone (8) chemotype in Egypt and Turkey varieties (77.2–86.2%) (Piras et al., 2013); trans-anethole (14) (38.3%) chemotype in Iranian N. sativa essential oil (Nickavar et al., 2003); p-cymene (9) (33%), and thymol (13) (26.8%) chemotype in Moroccan species (Moretti et al., 2004). The volatile oils from N. sativa seeds of Turkey and Egypt obtained by supercritical fractioned extraction with CO 2 also showed that thymoquinone was the major constituent (77.2–86.2%) followed by p-cymene (9) (5.4–11.0%) (Piras et al., 2013). An essential oil from Iranian N. sativa seeds was rich in thymoquinone (42.4%), p-cymene (14.1%) and caravacrol FIGURE 1 | Fatty acids in fixed oils from Nigella seeds. Frontiers in Pharmacology | www.frontiersin.org April 2021 | Volume 12 | Article 6253864 Salehi et al. A Comprehensive Review on Nigella Plants (10.3%) (Mahmoudvand et al., 2014). Figure 2 depicts the chemical structure of these phytochemicals and Table 2 the content of the majoronesandthesource. The essential oil composition is highly variable and probably more chemotypes exist. As an example, Jrah Harzallah et al. (2011) identified eighty-four compounds in the essential oil obtained by hydrodistillation of N. sativa seeds from Tunisia and the major one was the monoterpenes p-cymene (9) (49.48%), α-thujene (15) (18.93%), α-pinene (16) (5.44%), β-pinene (16) (4.31%). Alternatively, the bioactive compound thymoquinone represented only 0.79%. Similar results were found by Geng et al. (2009);N. sativa seeds contained p-cymene (9) (33.75%) as the major component using the hydrodestillation mode, with low content of thymoquinone (8) (3.73%), while using supercritical CO 2 extraction the major one was linoleic acid (1). Another study reported that N. sativa seeds from Tunisia and Morocco showed again that p-cymene (9) occurred in a higher relative concentration (60.5 and 56.7%, respectively) (Bourgou et al., 2010b;Badri et al., 2018). Furthermore, the essential oil extracted by hydrodistillation from Egyptian seeds showed the major components were p-cymene (9) (33.0%) and thymoquinone (8) (32.2%), followed by α-thujene (15) (13.0%) and camphene (19) (2.9%) (Viuda-Martos et al., 2011). Rarely, the composition of N. sativa seeds essential oil from Poland showed that two other monoterpenoids were present: cisand trans-4-methoxythujane (18) (Wajs et al., 2008). Bourgou et al. FIGURE 2 | Phytochemical components in essential oils from Nigella seeds. Frontiers in Pharmacology | www.frontiersin.org April 2021 | Volume 12 | Article 6253865 Salehi et al. A Comprehensive Review on Nigella Plants (2012) has identified also four terpenoids, trans/cis-sabinene hydrate methyl ether (20), 1,2-epoxy-menth-4-ene (21), and 1,2-epoxy-menth-4(8)-ene (22), in Tunisian N. sativa essential oil from seeds by nuclear magnetic resonance (Figure 2). Nigella damascena seed oil was characterized by almost 100% sesquiterpenes, of which β-elemene (17) (73.2%) was the most representative one (Moretti et al., 2004;Geng et al., 2009). Edris (2009) also remarked that this compound could be present even up to 73.0% in the essential oils of in N. orientalis and N. arvensis. Alternatively, N. arvensis could also contain other compounds in major levels, e.g. a methylated derivative of carvacrol (19) (26.4%), carvacrol methyl ether, followed by β-pinene (16) (21.4%). Alkaloids The bio-potential of nitrogen-containing heterocycles has been recently revised (Thakral and Singh, 2019). In plants, alkaloids contain one or more nitrogen atoms and usually are situated in some cyclic system. Similarly, these nitrogen-containing heterocycles, such as isoquinoline alkaloids and their N-oxides, can be a source of leads for drug discovery (Dembitskya et al., 2015). This included compound such as nigellimine (23) and nigellimine-N-oxide (24) from N. sativa seeds. This species also contains other type of alkaloids such as nigellicine (25), and nigellidine-4-O-sulfite (26) (Figure 3). Nigellidine (27) and its derivative methyl nigellidine, higenamine (28), and nigeglanine (29) were also characterized in N. sativa seeds (Atta Ur et al., 1995;Yun et al., 2014). Among them, methyl nigellidine, nigeglanoside and nigelloside could be used as markers to differentiate both species (Yun et al., 2014). Saponins Other studies also suggest the presence of phenolic compounds and triterpenes like saponins in the seeds of N. sativa and other Nigella species (Ali et al., 2008;Atta Ur, 1985;Atta Ur, 1992;El-Naggar et al., 2017;Kooti et al., 2016;Tiruppur Venkatachallam et al., 2010;Zribi et al., 2014). Saponins are a heterogeneous group of glycosides, which have one or more hydrophilic moieties combined with a lipophilic triterpene or steroid derivative. In particular, triterpene saponins are TABLE 2 | Main volatile compounds in essential oils from Nigella seeds. Compound name Source (origin) Relative amount (%) References Thymoquinone N. damascena (M), N. sativa seed (C), N. sativa seed (T), N. sativa seed (I), N. sativa seed (tk, E), N. sativa seed (ir), N. sativa seed (T), N. sativa seed (M) 0.1, 3.7, 3.0, 33.1–38.4 a , 77.2–86.2 b , 0.6, ND, 3.8 (Bourgou et al., 2010b;Geng et al., 2009;Jrah Harzallah et al. (2011);Moretti et al., 2004;(Piras et al., 2013) p-Cymene N. damascena (M), N. sativa seed (C), N. sativa seed (T), N. sativa seed (I), N. sativa seed (tk, E), N. sativa seed (ir), N. sativa seed (T), N. sativa seed (M) ND, 33.8, 60.5, ND, ND, 14.8, 49.5, 33.8 (Bourgou et al., 2010b;Geng et al., 2009;Jrah Harzallah et al. (2011);Moretti et al., 2004;(Piras et al., 2013) Dithymoquinone N. damascena (M), N. sativa seed (T), N. sativa seed (I), N. sativa seed (tk, E), N. sativa seed (ir), N. sativa seed (T), N. sativa seed (M) ND, ND, ND, ND, ND, ND (Bourgou et al., 2010b;Jrah Harzallah et al. (2011); Moretti et al., 2004;(Piras et al., 2013) Thymohydroquinone N. damascena (M), N. sativa seed (T), N. sativa seed (I), N. sativa seed (tk, E), N. sativa seed (ir), N. sativa seed (T), N. sativa seed (M) ND, 0.4, 1.1–2.3 a , ND, ND, ND (Bourgou et al., 2010b;Jrah Harzallah et al. (2011); Moretti et al., 2004;(Piras et al., 2013) Carvacrol N. damascena (M), N. sativa seed (T), N. sativa seed (I), N. sativa seed (tk, E), N. sativa seed (ir), N. sativa seed (T), N. sativa seed (M) ND, 2.4, 0.8–2.0 a , ND-7.9, 1.6, 0.6, ND (Bourgou et al., 2010b;Jrah Harzallah et al. (2011); Moretti et al., 2004;(Piras et al., 2013) Thymol N. damascena (M), N. sativa seed (T), N. sativa seed (I), N. sativa seed (tk, E), N. sativa seed (ir), N. sativa seed (T), N. sativa seed (M) ND, ND, 5.3–17.0 a , ND, ND, 26.8 (Bourgou et al., 2010b;Jrah Harzallah et al. (2011); Moretti et al., 2004;(Piras et al., 2013) α-Thujene N. damascena (M), N. sativa seed (T), N. sativa seed (I), N. sativa seed (tk, E), N. sativa seed (ir), N. sativa seed (T), N. sativa seed (M) ND, 6.9, ND, ND-0.4 b , 2.4, 18.9, 3.3 (Bourgou et al., 2010b;Jrah Harzallah et al. (2011); Moretti et al., 2004;(Piras et al., 2013) α-Pinene N. damascena (M), N. sativa seed (T), N. sativa seed (I), N. sativa seed (tk, E), N. sativa seed (ir), N. sativa seed (T), N. sativa seed (M) ND, 1.7, ND, ND, 1.2, 1.2, 5.4, 0.7 (Bourgou et al., 2010b;Jrah Harzallah et al. (2011); Moretti et al., 2004;(Piras et al., 2013) β-Pinene N. damascena (M), N. sativa seed (T), N. sativa seed (I), N. sativa seed (tk, E), N. sativa seed (ir), N. sativa seed (T), N. sativa seed (M) ND, 2.4, ND-0.4 a , ND, 1.3, 4.3, 1.1 (Bourgou et al., 2010b;Jrah Harzallah et al. (2011); Moretti et al., 2004;(Piras et al., 2013) γ-Terpinene N. damascena (M), N. sativa seed (T), N. sativa seed (I), N. sativa seed (tk, E), N. sativa seed (ir), N. sativa seed (T), N. sativa seed (M) ND, 3.5, 12.9–27.5 a , ND0.6 b , 0.5, 2.5, 2.4 (Bourgou et al., 2010b;Jrah Harzallah et al. (2011); Moretti et al., 2004;(Piras et al., 2013) trans-Anethole N. damascena (M), N. sativa seed (T), N. sativa seed (I), N. sativa seed (tk, E), N. sativa seed (ir), N. sativa seed (T), N. sativa seed (M) Tr, ND, ND, ND, 38.3 (Bourgou et al., 2010b;Jrah Harzallah et al. (2011); Moretti et al., 2004;(Piras et al., 2013) β-Elemene N. arvensis seed, N. damascena seed (M), N. sativa seed (M) 69.0, 73.2, 5.5 (Moretti et al., 2004;Edris, 2009) ND, Not detected/reported; C, China; I, India; Ir, Iran; E, Egypt; M, Morocco; T, Tunisia; Tk, Turkey; Tr, traces. a Depending on the extraction method and conditions. b Depending on the origin. Frontiers in Pharmacology | www.frontiersin.org April 2021 | Volume 12 | Article 6253866 Salehi et al. A Comprehensive Review on Nigella Plants FIGURE 3 | Example of chemical structures of alkaloids and saponins reported in N. sativa. Frontiers in Pharmacology | www.frontiersin.org April 2021 | Volume 12 | Article 6253867 Salehi et al. A Comprehensive Review on Nigella Plants highly characteristic compounds in Nigella seeds. Besides alphahederin (30) (Boubertakh et al., 2013), other relative saponins are: 3-O-[D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabi npyranosyl]-28-O-[-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyra nosyl-(1→6)-β-D-glucopyranosyl] hederagenin (nigella A) (31), 3-O-[D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arab inpyranosyl]-28-O-β-D-glucopyranosyl hederagenin (nigella B) (32), 3-O-[α-L-rhamnopyranosyl-(1→2)-α-L-arabinpyranosyl]-28-O- [β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl] hederagenin (nigella C) (33), 3-O-[D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl- (1→2)-α-L-arabinpyranosyl]-28-O-[-α-L-rhamnopyranosyl-(1→4)- β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl] hederagenin (nigella D) 34) (Figure 3). The latter were reported in the seeds of N. sativa and exhibit a broad spectrum of bioactivities (Chen et al., 2018;Dönmez and Mutlu, 2004). Others also found in the latter species are: 3-O-[L-rhamnopyranosyl-(1→2)- L-arabinopyranosyl]-hederagenin (kalopanaxsaponin A) (35) and 3-O-[-D-xylopyranosyl-(1→3)-L-rhamnopyranosyl-(1→2)- L-arabinopyranosyl]-hederagenin (kalopanaxsaponin I) (36) (Figure 3), with anticancer properties reported in vitro (Tian et al., 2006). Phenolic Compounds Concerning phenolic compounds, a recent study suggested that N. sativa,N. arvensis,N. damascena, and N. hispanica seeds have more flavonoids and phenolic acid derivatives than N. nigellastrum and N. orientalis seeds (Farag et al., 2014). Phenolic compounds are characterized to be small molecules with at least one phenol unit. Some common ones have been reported in the seeds of N. sativa seeds, e.g. kaempferol (37), quercetin (38), rutin (39), salicylic acid (40), p-hydroxybenzoic acid (41), methyl-4-hydroxybenzoate (42) and pyrogallol (43) (Xin et al., 2008;Boubertakh et al., 2013)(Figure 4). Table 3 shows examples of phenolic compounds described in Nigella plants and their contents. Kaempferol and quercetin derivatives are also common in other Nigella species (Farag et al., 2014). Moreover, characteristic glycosilated flavonoids, also found in the seeds, are: kaempferol 3-O-beta-D-galactopyranosyl-(1→3)-beta-D-glucopyranosyl(1→3)- beta-D-glucopyranoside (nigeglanoside) (44) (Hao et al., 1996), kaempferol 3-O-L-rhamnopyranosyl-(1→6)-O-[-D-glucopyranosyl (1→2)-O-D-galactopyranosyl (1→2)]-O-D-glucopyranoside (45) and kaempferol 3-O-D-glucopyranosyl-(1→2)-OD-galactopyranosyl-(1→2)-O-D-glucopyranoside 46) (Figure 4), which were reported in N. sativa (Liu et al., 2011;Xin et al., 2008)(Figure 4). The latter flavonoid is a taxonomic marker for distinguishing N. sativa from other Nigella species (Farag et al., 2014). Methanolic extracts from seeds, shoots and roots of Tunisian N. sativa contained phenolic compounds, including: gallic acid (47), p-hydroxybenzoic acid (41), vanillic acid (48), syringic acid (49), (–)-epicatechin (50), (+)-catechin (51), chlorogenic acid (52), p-coumaric acid (53), ferulic acid (54), trans-2-hydroxycinnamic acid (55), trans-cinnamic acid (56), quercetin (38), apigenin (57), and amentoflavone (58) (Figure 4). Nonetheless, among them, vanillic acid (48) was the major phenolic compound (Bourgou et al., 2008;Bourgou et al., 2010a). A recent study has shown that there are qualitative and quantitative differences between the phenolic compounds of ethanolic extracts from N. sativa and N. damascena seeds (Toma et al., 2015). For example, quercitrin (59) was detected in both extracts, hyperoside (60) and quercetin (38) in N. damascena, while kaempferol (37) was found only in N. sativa. Moreover, a recent study has reported a more complex phenolic profile, which was found complexed with N. damascena seeds proteins: gallic acid (47), protocatechuic acid (61), 2,5-dihydroxybenzoic acid (62), vanillic acid (48), (+)-catechin (51), caffeic acid (63), chlorogenic acid (52), syringic acid (49), (–)-epicatechin (50), p-coumaric acid (53), sinapic acid (64), hesperidin (65), quercetin 38) and kaempferol (37). Some of these compounds were also present in N. arvensis seeds (Alu’datt et al., 2016), while 7-methylkaempferol (rhamnocitrin) was also reported in the epigeal part of this species (Kirichenko et al., 1972). Other type of phenolic compounds reported in N. damascena seeds were hydroxytyrosol (66), 2,4-dihydroxyphenylacetic acid (67) and its methyl derivative (68), as well as a new phenolic ester, 1O-(2,4-dihydroxy) benzoylglycerol 69) (Fico et al., 2000) (Figure 4). Others Examples of other reported terpene chemicals include phytosterols such as β-sitosterol (70), Δ5-avenasterol (71), and Δ7-avenasterol (72), as well as stanols such as cycloartenol (73) (Figure 5). Other triterpenes like β-amyrin (74) and butyrospermol (75) (Ramadan and Mörsel, 2002;Ahmad et al., 2013;Ijaz et al., 2017), as well as α-tocopherol (76), γ-tocopherol (77), and β-carotene (78) are also found in seeds (Ramadan and Mörsel, 2004)(Figure 5). Factors that Affect the Phytochemical Composition Phytoconstituents of Nigella may vary even within the same species and this is related to many factors such as growing and climatic conditions, location, different organs of the plants and the extraction methods used (Edris, 2010;Manju et al., 2016;Saxena et al., 2017) (Tables 2 and 3). Secondary metabolites, compounds which are not directly related to the development, growth and reproduction of plantsbuttheyhavesignificant performance in chemical communication, primary defense against biotic and abiotic stress (Sarkar and Shetty, 2014) and also in epigenetic memory. The chemical composition may vary also during development stage and this was confirmed by Zribi et al. (2014) who demonstrated that the total phenolics, flavonoids, flavonols and flavones, alkaloids and proanthocyanidins contents of Tunisian and Indian N. sativa aqueous extracts were the highest in the vegetative stage. Moreover, the aerial parts from the two varieties were richer in total phenolics and flavonoids, including flavonols, flavones and proanthocyanidins, than seeds. Salinity is also another factor that greatly influenced the phenolic composition of Nigella seeds; e.g. generally the content of certain phenolic compounds decreased, including the major one vanillic acid (45), while the content of trans-cinnamic acid (53), Frontiers in Pharmacology | www.frontiersin.org April 2021 | Volume 12 | Article 6253868 Salehi et al. A Comprehensive Review on Nigella Plants FIGURE 4 | Example of chemical structures of phenolic compouds reported in N. glandulifera,N. sativa,N. damascena, and N. arvensis. Frontiers in Pharmacology | www.frontiersin.org April 2021 | Volume 12 | Article 6253869 Salehi et al. A Comprehensive Review on Nigella Plants TABLE 4 | Clinical trials on effect of Nigella sativa to various system disorders, diseases and conditions. Intervention Application, duration Type of study Number of patients/study design a Control Main effects a References Powdered N. sativa seed Oral application for 8 weeks, 2 g of powder per day Double-blind placebo controlled randomized clinical trial 40 patients with HT (aged between 22 and 50 years) Placebo (starch) ↓body weight and BMI, ↓ TSH and anti-TPO antibodies, ↑T3, ↓serum VEGF concentration (Farhangi et al., 2016) N. sativa powder Oral application for eight weeks Randomized double-blind trial 40 patients with HT, 22–50 years Placebo (starch) ↓serum IL-23, ↓TSH and anti-TPO antibodies, ↑ serum T3, ↓body weight (Tajmiri et al., 2016) N. sativa seed powder Oral application for 8 weeks, 2 g per day Double-blind placebo controlled randomized clinical trial 40 patients with HT (aged between 22 and 50 years) Placebo (starch) ↓serum LDL and T3, ↑HDL (Farhangi et al., 2018) N. sativa powdered seed Oral application of 500 mg in combination with 500 mg of metformin, 10 mg atorvastatine, 150 mg aspirin Randomized clinical trial 80 patients with metabolic syndrome and poor glycemic control (HbA1C>7%) 500 mg of metformin, 10 mg atorvastatine, 150 mg aspirin ↓FBG, PPBG, HbA1c, LDL (Najmi et al., 2012) N. sativa seed oil Oral application for 3 months, 2.5 mL two times daily Double-blind placebo controlled randomized clinical trial 70 patients with type II diabetes Mineral oil ↓FBG, PPBG, HbA1c (Hosseini et al., 2013) N. sativa seed powder Oral application of 2g powder daily, for one year in addition to their standard medications Double-blind placebo controlled randomized clinical trial 114 patients with type 2 diabetes on standard oral hypoglycemic drugs Placebo (charcoal) ↓FBG, HbA1c, TBARS ↑ TAC, SOD, GSH (Kaatabi et al., 2015) N. sativa oil soft gel capsules Oral application of 3g oil daily, for 12 weeks Double-blind placebo controlled randomized clinical trial 72 patients with diabetes type 2 Sunflower oil gel capsules ↓FBG, HbA1c, TG, LDL (Heshmati et al., 2015) N. sativa seed oil Oral application of 2.5 mL two times daily for two months Double-blind placebo controlled randomized clinical trial 68 healthy men 20–45 years of age with infertility lasting more than one year Liquid paraffin Sperm count, motility, morphology and semen volume, pH and round cells were improved significantly (Kolahdooz et al., 2014) N. sativa seed extract Two test groups received 100 and 200 mg of extract twice a day for 8 weeks Double-blind placebo controlled randomized clinical trial 119 healthy male volunteers, aged 35 to 50 Placebo ↓systolic and diastolic BP in a dose-dependent manner (Dehkordi and Kamkhah, 2008) N. sativa seed oil Oral application of 2.5 mL oil two times per day for 8 weeks Randomized, double-blind placebocontrolled trial 70 healthy volunteers aged 34–63 years Mineral oil ↓systolic and diastolic BPs (Fallah Huseini et al., 2013) N. sativa oil 6 mg/kg daily, for 30 days Prospective and double-blind clinical study 66 patients with allergic rhinitis Placebo ↓nasal mucosal congestion, nasal itching, sneezing attack, runny nose, turbinate hypertrophy, and mucosal pallor during the first 2 weeks of the study (Nikakhlagh et al., 2011) N. sativa nasal spray 2 puffs/day of N. sativa nasal spray (1 g/day of N. sativa) for 8 weeks Randomized double-blind placebocontrolled trial 65 patients with mild to moderate chronic rhinosinusitis Placebo (2 puffs/day of sodium chloride spray 0.65%) Lund–McKay, lund Kennedy, and Sino-nasal outcome Test-22 scores significantly decreased in the intervention group (Rezaeian and Amoushahi Khouzani, 2018) N. sativa oil Topical application, twice a day for 6 months Randomized, double-blind clinical trial 52 patients with vitiligo lesions Fish oil Reduction in size of lesions (Ghorbanibirgani et al., 2014) N. sativa ointment (2%) Ointment (1 G) topically applied on eczematous lesions twice a day for a period of 4 weeks Randomized double-blind placebocontrolled trial 60 patients with hand eczema, 18–60 years Betamethasone and eucerin ↓Dermatology life quality index score in Nigella and betamethasone groups compared to eucerin (Yousefiet al., 2013) (Continued on following page) Frontiers in Pharmacology | www.frontiersin.org April 2021 | Volume 12 | Article 62538616 Salehi et al. A Comprehensive Review on Nigella Plants positive, e.g. in total cholesterol and asthma, and subject of publications (Qidwai et al., 2009;Koshak et al., 2017). Furthermore, in the following section the results of other clinical studies regarding preventive and relieving effects of N. sativa on metabolic disorders and risk factors (diabetes, obesity and hypertension), as well as other effects are shown. In Table 4, we have summarized these effects. The effects of other Nigella plants are also commented. Effect of N. sativa Supplementation on Patients with Metabolic Disorders and Risk Factors Metabolic Syndrome The possibility of application of N. sativa seed in patients with metabolic syndrome was firstly investigated by Najmi et al. (2008). They found that the administration with N. sativa seed oil (2.5 mL twice a day for 6 weeks) in patients with metabolic syndrome significantly decreased FBG and LDL and increased HDL levels. The same group of authors (Najmi et al., 2012) analyzed the effect of supplementation with powdered N. sativa seed (500 mg/day for two months) in patients with low glycemic control (glycate hemoglobin—HbA1C was lower than 7%). As a result, in the intervention group significant lowering of FBG, postprandial blood glucose (PPBG) and HbA1c was observed. Conversely, a recent cross-over study (2 months treatment, 2 weeks washout period) evaluated the effects of N. sativa,asa form of traditional bread spiked with seeds (2 g), on FBG, BP and anthropometric indices (BW, waist circumference or WC, and BMI body mass index) in patients with metabolic syndrome. It found no significant effect between the mean of changes of TABLE 4 | (Continued) Clinical trials on effect of Nigella sativa to various system disorders, diseases and conditions. Intervention Application, duration Type of study Number of patients/study design a Control Main effects a References N. sativa seed powder and ointment 12 weeks, group I: 10% w/ w ointment with N. sativa oil extract; group II: capsules with 500 mg of N. sativa powder, three times daily; group III: Combination of ointment and capsules Randomized clinical trial 60 patients with mild to moderate plaque and palmoplanter psoriasis - Group I—total healing of psoriatic lesions, with good response in 65% of patients, and a relapse rate of 31% four weeks after cessation of treatment; group II—good response in 50% of patients, with a relapse rate of 50% observed four weeks after application; group III—total cure of lesions, and good responses in 85% of patients, with a relapse rate of 18% (Jawad et al., 2014) N. sativa oil Two placebo capsules daily for 1 month, followed by a month of NS oil capsules 500 mg twice per day Placebo controlled clinical trial 40 female atients with rheumatoid artritis Placebo (two starch capsules per day) ↓disease activity score, ↓ number of swollen joints and the duration of morning stiffness (Gheita and Kenawy, 2012) N. sativa oil 500 mg oil capsules two times daily for 8 weeks Randomized, double-blind, placebocontrolled clinical trial 42 patients with rheumatoid artritis Placebo ↑IL-10, ↓MDA, ↓NO (Hadi et al., 2016) N. sativa oil Topical application twice a day (in the morning and night) for 21 days Double-blind, parallel, clinical trial 52 pateints with osteoarthritis, 60–80 years Diclofenac gel Better pain relief effect compared to diclofenac gel according to KOOS score (38.88 ±17.84 and 50.33 ± 20.38, respectively) (Azizi et al., 2019) N. sativa seed Oral administration: 500 mg in capsules (twice a day for 9 weeks) Randomized study 20 healthy humans Placebo: Psyllium seed husk in capsuels Improvement in the parameters studied: Score of logical memory tests, attention test (letter cancenlation test and trail making test), cognitive test (Scroop)etc. (Bin Sayeed et al., 2013) a BMI, body mass index; BP, blood pressure; FBG, fasting blood glucose; HbA1c, hemoglobin A1c; HT, Hashimoto’s thyroiditis; IL, interleukin; LDL, low density lipoprotein; MDA, malondialdehyde; PPBG, postprandial blood glucose; T3, total triiodothyronine; TAC, total antioxidant capacity; TBARS, thiobarbituric acid reactive substances; TG, triglycerides; TPO, thyroid peroxidase; TSH, thyroid-stimulating hormone; VEGF, vascular endothelial growth factor. Frontiers in Pharmacology | www.frontiersin.org April 2021 | Volume 12 | Article 62538617 Salehi et al. A Comprehensive Review on Nigella Plants parameters in the beginning and end of study (time effect), with the exception of diastolic BP (Mohtashami, 2019). Diabetes Oxidation and inflammation are important factors connected with occurrence of various chronic diseases, such as diabetes mellitus type 2 (Pouvreau et al., 2018). Due to the antioxidant, antiobesity and antidiabetic effects of N. sativa seeds and their constituents, this plant species can offer potential in prevention and treatment of type 2 diabetes. A recent review suggests that N. sativa can improve glycemic stages and lipid profile in diabetes (Heshmati and Namazi, 2015). Hosseini et al. (2013) investigated anti-hyperglycemic effect of N. sativa seed oil in type II diabetic patients. As a result of supplementation with 5 mL of oil/day for three months, blood levels of fasting and 2 h PPBG, as well as HbA1c were significantly decreased compared to placebo group. This hypoglycemic effect can be result of an insulin senzitation and stimulation of pancreatic beta-cell function, resulting in intensified activity and consequent decrease in glucose level. Moreover, Kaatabi et al. (2015) reported pronounced antidiabetic activity after three-month application of N. sativa powdered seed (2 g/day) in combination with oral hypoglycemic agent in patients with type 2 diabetes. In this study, N. sativa received group showed significant reduction of FBG, HbA1C, and TBARS, and at the same time noticeable increase of the total antioxidant capacity, SOD, and glutathione levels were recorded in the intervention group. Heshmati et al. (2015) investigated effect of N. sativa seed oil application (3 g/day) on glucose metabolism and lipid concentrations in patients with type 2 diabetes. According to this study, FBG, HbA1C, and levels of triglycerides (TG) and LDL significantly changed in the intervention group compared to the placebo one. On the other hand, insulin level and its resistance decreased and HDL increased in the intervention group, but after adjusting for confounder factors, these parameters were not significant. Overall, the potential antidiabetic mechanisms of N. sativa could be mediated through a change in the oxidative status (either via upregulation of endogenous antioxidants or reduction of oxidative species), reduction of inflammation, and improvement of lipid profiles (Yimer et al., 2019). Furthermore, a recent meta-analysis confirms that supplementation with N. sativa could be a suitable choice to manage the complications of type 2 diabetes, including FBS (−17.84 mg/dL), HbA1c (−0.71%), total cholesterol (−22.99 mg/dL), and LDL (−22.38 mg/dL) (Daryabeygi-Khotbehsara et al., 2017). Hypercholesterolemia and Obesity Nigella sativa powder supplementation (1 g/day) for sixty days caused momentous reductions in concentrations of LDL, TG levels and enhancement in HDL level in hypercholesterolemic patients (Tasawar et al., 2011). Moreover, a double-blind, randomized, placebo-controlled 4-weeks trial showed that the administration of N. sativa seeds (1g/day) can reduce total cholesterol and LDL. Thus, it can be interesting as lowering lipid agent in hyperlipidaemic subjects (Pelegrin et al., 2019). Nigella sativa oil (3 g/day for 8 weeks) was also tested in a lowcalorie diet on cardiometabolic risk factors in obese women. Compared to the placebo group, in the N. sativa treated group, weight (−6.0%) and WC (−6.9%) decreased. It also favored a reduction in triglyceride and LDL levels (Mahdavi et al., 2015). Recently, a recent meta-analysis (literature till June 2017) has been performed on the effects of supplementation with N. sativa on some anthropometric indices in adult subjects. It indicated that N. sativa supplementation exerts a moderate effect on reduction in BW (−2.11 kg), BMI (−1.16 kg/m 2 ) and WC (−3.52 cm) (Namazi et al., 2018). Nonetheless, other metaanalysis performed till January 2018 suggested that N. sativa supplementation have only an effect on BW (−1.76 kg) and BMI (−0.85 kg/m 2 ) in adults compared to placebo (Mousavi et al., 2018). Hypertension There are several clinical studies reporting positive effect of N. sativa application on hypertension. Dehkordi and Kamkhah (2008) demonstrated that application of N. sativa seeds extract (100 and 200 mg for eight weeks) in patients with mild hypertension led to significant reduction of systolic and diastolic BP compared to placebo. At the same time, a significant decrease in total and LDL cholesterol was observed and no other complications caused by the treatment were found. Similar results were obtained for N. sativa seed oil. Fallah Huseini et al. (2013) exhibited that its application in a dose 2.5 mL two times per day for 8 weeks reduced systolic and dyastolic BP in healthy volunteers. Authors suggested that the exhibited effect could be attributed to the activity of thymoquinone (8), one of main constituents of the volatile oil from N. sativa seed, as commented before. Some of the cardiovascular benefits evidenced in vitro and now in vivo through risk factors, such as blood lipids, and BP, could be related to the presence of omega-6 fats as linoleic acid, which is the major fatty acid of the seed oil. Nonetheless, the cardiovascular health benefits of linoleic acid are controversial (Hooper et al., 2018;Marklund et al., 2019), and the positive effects of minor phytochemicals remains unclear. Other Effects of N. sativa Effect of N. sativa Supplementation in Patients with Hashimoto’s Thyroiditis (HT) HT is one of the most common human autoimmune diseases influencing the thyroid glands and an organ-specific T-cell mediated disease (Chistiakov, 2005). The disease is ten times more frequent in women than in men and it affects 2% of general population. HT is associated with serious alterations in composition and the transport of lipoproteins. Several clinical studies have demonstrated the efficacy of N. sativa application in patients with HT. According to the study conducted by Farhangi et al. (2016), the application of powdered N. sativa seeds (2 g) for eight weeks significantly decreased BW and BMI compared to placebo. Also, a positive effect has been achieved on thyroid function. A decrease in the serum concentrations of thyroid stimulating hormone (TSH) and anti-thyroid peroxidase (antiTPO) has been shown, while serum concentrations of T3 increased in N. sativa treated group. In the same study, significant reduction of vascular endothelial growth factor Frontiers in Pharmacology | www.frontiersin.org April 2021 | Volume 12 | Article 62538618 Salehi et al. A Comprehensive Review on Nigella Plants (VEGF) was noted in the intervention group. It has been observed that its concentration grows during pathological states characterized by increased TSH secretion (Farhangi et al., 2016). These results were confirmed by Tajmiri et al. (2016) and Farhangi et al. (2018), who showed lower BMIs in the group of patients with HT after the treatment with N. sativa seeds. This positive effect was exhibited through hypolipidemic effect: lowering serum LDL cholesterol and TG concentrations, while raising the level of HDL cholesterol. Beside this effect, Tajmiri et al. (2016) showed that treatment with N. sativa seed powder for eight weeks reduced levels of serum TSH, anti-TPO and IL-23, while level of serum T3 was increased. Effect of N. sativa Supplementation on Patients with Reproductive System Disorders Kolahdooz et al. (2014) studied effect of N. sativa seed oil on abnormal semen quality in infertile man. These authors showed that the daily application of 5 mL for two months significantly improved sperm count, morphology and motility and volume of semen, pH, and number of round cells. The exact mechanism of the exhibited effect is not determined, but it is likely related to strong antioxidant activity of the oil. Namely, it is well known that high level of oxidative stress contributes to decreased semen quality (Schulte et al., 2010). Effect of N. sativa Supplementation on Patients with Allergic Rhinitis The potential of application of N. sativa seeds and its oil in patients with allergic rhinitis has been also confirmed in clinical studies. Nikakhlagh et al. (2011) showed in prospective and double blind clinical trial that the oral administration of N. sativa seed oil (6 mg/kg daily) for 30 days in patients with allergic rhinitis significanly decreased the severity of respiratory symptoms, such as the presence of the nasal mucosal congestion, nasal itching, runny nose, sneezing attacks, turbinate hypertrophy, and mucosal pallor. Moreover, Rezaeian and Amoushahi Khouzani (2018) investigated the effect of N. sativa nasal spray in randomized clinical study. In this study patients in the intervention group received 2 puffs/day of N. sativa nasal spray (i.e. 1 g/day of N. sativa) and in the placebo group received 2 puffs/day of sodium chloride spray (0.65%). Lund–McKay, Lund Kennedy, and Sino-Nasal Outcome Test-22 scores were used and the outcomes were significantly lower in the intervention group compared to the placebo group. These findings are in line with previous reports about the antihistamine properties of N. sativa (Boskabady and Sheiravi, 2002;Kanter et al., 2006). Effect of N. sativa Supplementation on Patients with Skin Diseases Several clinical trials demonstrated positive effects of N. sativa application in resolving symptoms related to some skin diseases. Ghorbanibirgani et al. (2014) showed positive effects of 6 months topical application of N. sativa oil on vitiligo lesions compared to fish oil according to Vitiligo Area Scoring Index. In another study, Yousefiet al. (2013) investigated the therapeutic potential of 2% ointment topically applied (twice/day during 4 weeks) in patients with hand eczema compared to eucerin and betamethasone. According to scores obtained using Hand Eczema Severity index and Dermatology Life Quality Index, they showed that N. sativa ointment could have the same efficacy as betamethasone, while it is more efficient compared to eucerin. Jawad et al. (2014) investigated effect of various preparations of N. sativa in psoriasis treatment. A first group has been receiving Nigella ointment topically (10% w/w, twice daily), the second group took crude pulverized plant (capsules with 500 mg three times daily), while the third group received the combination of both treatments. The inspected reaction has been estimated following the Psoriasis Area and Severity Index score, while MDA serum level was used as indicator of the oxidative stress. After 12 weeks of treatment ointment reached total healing of psoriatic lesions, with good response in 65% of patients, and a relapse rate of 31% four weeks after cessation of treatment. Oral doses of N. sativa developed good response in 50% of patients, with a relapse rate of 50% observed four weeks after application. The combination of ointment and oral doses gained best results—total cure of lesions, and good responses in 85% of patients, with a relapse rate of 18%. All of these positive effects of N. sativa in dermatological diseases could be attributed to its antimicrobial, antioxidant, immunomodulatory, and anti-inflammatory effects. Effect of Nigella sativa Supplementation in Patients with Rheumatic Artritis The efficacy of black cumin oil in patients with rheumatoid arthritis (RA) was evaluated in female patients diagnosed with RA. Application of N. sativa oil in capsules (500 mg) twice daily exhibited improvement in disease activity score compared to placebo. Respectively, a pronounced improvement was demonstrated in amount of inflamed joints and occurrence of morning stiffness (Gheita and Kenawy, 2012). These results were confirmed by Hadi et al. (2016) with higher doses of N. sativa oil (1 g of oil/day, divided in two capsules). They showed that after 8 weeks of application in patients with RA noticeable decrease of serum MDA and NO was observed in the intervention group, while at the same time the serum level of anti-inflammatory cytokine IL-10 was increased (Hadi et al., 2016). Azizi et al. (2019) compared the effects of topical application of N. sativa oil and diclofenac gel in patients with osteoartritis, one of the most common diseases in aging population. In this study, pain score has been expressed as average number obtained using Knee injury and Osteoarthritis Outcome Score scale. It has been observed that first ten days there has been no significant differences in placebo and the intervention group. Still, after 21 days of application better pain relief effect was observed after treatment with N. sativa oil compared to diclofenac gel. Effects on Memory, Attention and Cognition In a randomized study with 20 healthy humans, the effects of 500 mg of N. sativa seeds (twice a day for 9 weeks) on memory, attention, and cognition were studied. The results showed an improvement in the score of the tests studied, without an Frontiers in Pharmacology | www.frontiersin.org April 2021 | Volume 12 | Article 62538619 Salehi et al. A Comprehensive Review on Nigella Plants alteration of the biochemical markers of cardiac, liver and kidney functions (Bin Sayeed et al., 2013). Effects of Other Nigella Species Among other herbs, there are several studies on the clinical use of N. ciliaris during childbirth and postpartum (Ali-Shtayeh et al., 2015), against diabetes (Ali-Shtayeh et al., 2012) and hypertension (Ali-Shtayeh et al., 2013), as well as N. arvensis against psoriasis (Jaradat et al., 2016b) and cancer (Jaradat et al., 2016a), which were performed in Palestine using questionnaires. Some patients seem to be satisfied with the outcomes of herbal therapy (Ali-Shtayeh et al., 2012;Ali-Shtayeh et al., 2013), but the authors did not reveal the herb treatment that was successful to manage the latter diseases/conditions. In any case, clinical evidences are still lacking and no studies have been found in the abovementioned database https://clinicaltrials.gov/. CONCLUSION Nigella seeds and extracts have a wide range of bioactivities, which have been demonstrated in vitro and in vivo. Nonetheless, N. sativa is the most studied species probably due to their popularity in folklore medicine. In all cases, future perspectives should be oriented to perform a better characterization of the constituents of the extracts since most studies were only focused on determining the concentration of thymoquinone, but other phytochemicals could be present as in this review more than 80 compounds have been reported. This is also important to remark that bioavailability could affect the way that Nigella components act in vivo, e.g., some studies administered Nigella products orally, while other intravenously. When referring to clinical trials, it seems that the supplementation of N. sativa can exert some effects on diabetes, obesity, and hypertension, among others. Nonetheless, the administration way (seeds, powder, oil, etc.), the dose and the treatment period are highly variable and should be further established from a therapeutic point of view, as well the chemical and phytochemical composition. This should be further study in order to formulate functional ingredients/nutraceuticals to promote the health benefit and how should be added into food. Moreover, besides the pleiotropic health applications of Nigella,it can be useful in nanotechnology applications as well as to modulate the activity of other therapeutic agents. It is important to highlight that the culinary and medicinal use of some Nigella seeds as spice, condiment or infusion (e.g.,N. sativa,N. damascena, and N. arvensis) suggests that their consumption is safe. The seeds are characterized by a low toxicity degree (Ali and Blunden, 2003) and no serious side effects in clinical trials (Namazi et al., 2018), but more studies were focused on N. sativa. In fact, toxicity studies performed in animal models also suggest a wide of margin of safety for using N. sativa fixed oil, with high LD 50 value (28.8 ml of oil/kg BW administered orally to mice) (Zaoui et al., 2002), in powder form (Dollah et al., 2013) and as extracts (Vahdati-Mashhadian et al., 2005). AUTHOR CONTRIBUTIONS All authors contributed equally to the manuscript. Conceptualization, BS, CQ, MI, IU-H, Jˇ Z,IA-R,SS,YT,KA,HA, AS-C,DM,GS,MM,US,RK,HS;validationinvestigation—data curation writing—all authors; review and editing, MdMC, AR, and JS-R. 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